Heterochromatin was originally defined as loci of nuclear DNA that remain highly condensed during interphase. Faithful chromosome segregation and genome stability requires heterochromatin, demonstrating the critical importance of assembling these loci; in fact, knockout mice for Suv39h, a component required for heterochromatin formation, have increased tumor risks. Recent data indicates that the RNAi machinery is required for heterochromatization by an as yet unknown mechanism. We have designed experiments that will address this problem in the genetically tractable fission yeast, S. pombe. We intend to determine (1) whether RNAi mediates heterochromatin formation in cis or in trans or both, (2) whether heterochromatin targeting requires transcription of the target locus, (3) whether RNAi-mediated heterochromatin formation is heritable in the absence of dsRNA, (4) whether RNAi-mediated heterochromatin formation results in heterochromatin spreading, and (5) the order of assembly of components of the RNAi and heterochromatin-specifying machinery. Furthermore, we have designed a novel genetic screen to isolate novel factors that are both required for and disrupt RNAi-mediated heterochromatin formation. [unreadable] [unreadable]